1. Field of the Invention
This application is directed to methods for the enzymatic synthesis of .alpha.-sialylated oligosaccharides. Specifically, in the methods of this invention .alpha.2,3-sialyltransferase is employed to transfer sialic acid or an analogue thereof, employed as its CMP-nucleotide, to the non-reducing terminus of an oligosaccharide which oligosaccharide has a fucosyl group in the position penultimate to the non-reducing sugar terminus of the oligosaccharide.
2. References
The following references are cited in this application as superscript numbers at the relevant portion of the application and are incorporated herein in their entirety.
1. Horowitz, The Glycoconjugates, Vols. I-V, Pigman, Editor New York Academic Press (1977, 1978, 1982, 1983) PA0 2. Hakomori, Adv. Cancer Res., 52:257-331 (1989) PA0 3. Venot et al., U.S. Pat. No. 5,352,670 PA0 4. Schengrund et al., J. Biol. Chem., 264: 13233-13237 (1989) PA0 5. Paulson, "Interaction of Animal Viruses with Cell Surface Receptors" in The Receptors Conn Ed N.Y. Acad. Press, pp. 131-219 (1985) PA0 6. Feizi, TIBS, 16:84-86 (1991) PA0 7. Brandley et al., Cell, 63:861-863 (1990) PA0 8. Houghton et al., Symposium on Gangliosides and Cancer, pp. 233-237, VCH Publishers (1988) PA0 9. Irie et al., Symposium on Gangliosides and Cancer, pp. 247-257, VCH Publishers (1988) PA0 10. Howard, in "Towards Better Carbohydrate Vaccines"; Proceedings of a meeting organized by the World Health Organization, R. Bell, G. Torrigani, Editors, pp. 212-236, Wiley, Chichester (1987). PA0 11. Henningsson et al., Cancer Immunol. Immunother., 25:231-241 (1987) PA0 12. Fung et al., Cancer Res., 50:4308-4314 (1990) PA0 13. Livingston et al., Proc. Natl. Acad. Sci. (USA), 84:2911-2915 (1987) PA0 14. Naor et al., Prog. Allergy, 22:107-146 (1977) PA0 15. Orskov et al., J. Exp. Med., 149:669-685 (1979) PA0 16. Barsoum et al., Mol. Immunol., 18:495-550 (1981) PA0 17. Jennings et al., U.S. Pat. No. 4,727,136 (1985) PA0 18. Honda et al., J. Biochem., (Tokyo) 95:1323-1329 (1984) PA0 19. Nakamura et al., J. Biochem., (Tokyo) 99:219-226 (1986) PA0 20. Reuter et al., Glycoconjugate J., 5:133-135 (1988) PA0 21. Weinstein et al., J. Biol. Chem., 257:13835-13844 (1982) PA0 22. Paulsen et al., J. Biol. Chem., 252:2356-2362 (1977) PA0 23. Evans-Sadler et al., J. Biol. Chem., 254:4434-4443 (1979) PA0 24. Conradt et al., Japanese-German Symp. pp. 104-105 Berlin (1988) PA0 25. Joziasse et al., J. Biol. Chem., 260:4941-4951 (1985) PA0 26. Evans-Sadler, J. Biol. Chem., 254:5934-5941 (1979) PA0 27. Bergh et al., Eur. J. Biochem., 136:113-118 (1983) PA0 28. Higa et al., J. Biol. Chem., 260:8838-8849 (1985) PA0 29. Paulsen et al., Eur. J. Biochem., 140:523-530 (1984) PA0 30. de Heij et al., Carbohydr. Res., 149:85-99 (1986) PA0 31. Sialic Acids in "Cell Biology Monographs" Schauer, Editor, Vol. 10 (1982) PA0 32. Okamoto et al., Tetrahedron, 46, No. 17, pp. 5835-5837 (1990). PA0 33. Ratcliffe et al., U.S. Pat. No. 5,079,353, (1987). PA0 34. Abbas et al., Proc. Japanese-German Symp. Berlin, pp. 20-21 (1988). PA0 35. Paulsen, Agnew. Chem. Int. Ed. Eng., 21:155-173 (1982). PA0 36. Schmidt, Agnew. Chem. Int. Ed. Eng., 25:212-235 (1986). PA0 37. Fugedi et al., Glycoconj. J., 4:97-108 (1987). PA0 38. Kameyama et al., Carbohydr. Res., 209:C.sub.1-C.sub.4 (1991). PA0 39. Brossmer et al., Biochem. Biophys. Acta., 96:1282-1289 (1980) PA0 40. Gross et al., Eur. J. Biochem., 168:595-602 (1987) PA0 41. Zbiral et al., Monatsh. Chem., 119:127-141 (1988) PA0 42. Sharma et al., Carbohydr. Res., 175:25-34 (1989) PA0 43. Hasegawa et al., J. Carbohydr. Chem., 8:579-588 (1989) PA0 44. Zbiral et al., Monatsh. Chem., 116:87-98 (1985) PA0 45. Salunkhe et al., Liebigs Ann. Chem., pp. 187-189 (1988) PA0 46. Hasegawa et al., J. Carbohydr. Chem., 8:135-144 (1989) PA0 47. Gross et al., Biochemistry, 27:4279-4283 (1989) PA0 48. Zbiral et al., Carbohydr. Res., 194:C15-C18 (1989) PA0 49. Gross et al., FEBS Lett., 232:145-147 (1988) PA0 50. Paulsen et al., Liebigs Ann. Chem., pp. 277-279 (1988) PA0 51. Nakajima et al., Agric. Biol. Chem., 52:1209-1215 (1988) PA0 52. Baumberger et al., Helv. Chem. Acta, 69:1535-1541 (1986) PA0 53. Beau et al., Eur. J. Biochem., 160:531-540 (1980) PA0 54. Mack et al., Tetrahedron Lett., 28:191-194 (1987) PA0 55. Gross et al., Eur. J. Biochem., 177:583-589 (1988) PA0 56. Christian et al., Carbohydr. Res., 194:49-61 (1989) PA0 57. Conradt et al., FEBS Lett., 170:295-300 (1984) PA0 58. Christian et al., Carbohydr. Res., 162:1-11 (1987) PA0 59. Haverkamp et al., Hoppe-Seyler's Z. Physiol. Chem., 360:159-166 (1979) PA0 60. Gross et al., Glycoconj. J., 4:145-156 (1987) PA0 61. Hagedorn et al., XIIIth Carbohydr. Symp., Ithaca (1986) A4 PA0 62. Toone et al., Tetrahedron, No. 17, 45:5365-5422 (1989) PA0 63. Palcic, Methods in Enzymology 230:300 (1994) PA0 64. Beyer et al., Adv. Enzymol., 52:24-158 (1981) PA0 65. International Patent Application Publication No. WO97/18302 PA0 66. Le et al., J. Chromatography 781:515-522 (1997) PA0 67. Kashem et al., U.S. Pat. No. 5,374,655 PA0 68. Gokhale et al., Can J. Chem. 68:1063 (1990) PA0 69. Srivastava et al., J. Biol. Chem. 267:22356-22361 (1992) PA0 70. Stangier et al., Carbohydrate Res. 305:511-515 (1998) PA0 71. Baisch et al., Bioorganic and Medicinal Chemistry Letters 6:2953-2956 (1996) PA0 .alpha.Neu5Ac(2-3).beta.Gal(1.fwdarw.3/4).beta.GlcNAc-.sup.21 PA0 .alpha.Neu5Ac(2-6).beta.Gal(1-4).beta.GlcNAc-.sup.21,22 PA0 .alpha.Neu5Ac(2-3).beta.Gal(1-3).alpha.GalNAc-.sup.23-25 PA0 .alpha.Neu5Ac(2-6).alpha.GalNAc-.sup.26,28 PA0 .alpha.Neu5Ac(2-6).beta.GlcNAc-.sup.29,30.
3. State of the Art
Carbohydrates and/or oligosaccharides are present on a variety of natural and pathological glycoconjugates.sup.1. Of particular interest are carbohydrates and oligosaccharides containing sialic acid residues particularly at the nonreducing sugar terminus.sup.31 Such sialic acid terminated carbohydrates and oligosaccharides are present in a number of products which have been implicated in a wide range of biological phenomena based, in part, on the concept of recognition signals carried by the carbohydrate structures and by their binding to specific ligands.
Specifically, such sialic acid terminated carbohydrates and oligosaccharides are believed to be receptors for the binding of toxins.sup.4, pathogenic agents such as viruses.sup.5, and are believed to be recognition sites for a variety of lectins, particularly those involved in cellular adhesion.sup.6,7, etc.
Similarly, certain oligosaccharides including sialic acid terminated oligosaccharides have been identified as capable of suppressing a cell-mediated immune response to an antigen. The ability of such oligosaccharides to suppress a cell mediated immune response to an antigen is described by Venot et al..sup.3
Additionally, the presence of certain sialyl terminated oligosaccharides in tumor-related antigens is documented in the art.sup.1 and, in general, the structures of the oligosaccharides present on such antigens have been modified in some way from normal oligosaccharides so as to lead to the expression of tumor related antigens.sup.2. The prospect of passive immunotherapy with monoclonal antibodies directed against some sialylated tumor-associated antigens, such as the gangliosides GD.sub.2, GD.sub.3 and GM.sub.2, in patients with melanoma has been investigated.sup.8,9.
The synthesis of such oligosaccharides often involves complex chemical reactions with corresponding low yields. Accordingly, there has been much interest in using glycosyltransferases in synthesizing at least a part of these molecules.
Glycosyltransferases are a highly polymorphic group of membrane-bound enzymes of endoplasmic reticulum and Golgi bodies that catalyze the transfer of a single monosaccharide unit from a nucleotide donor to the hydroxyl group of an acceptor saccharide in the biosynthesis of N-glycan (Asn-GlcNAc N-glycosidic linkage; GlcNAc, N-acetylglucosamine) and O-glycan (Ser/Thr-GalNAc, O-glycosidic linkage; GalNAc, N-acetylgalactosamine) moieties of glycoproteins and glycolipids.
The eukaryotic sialyltransferases comprise a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid (NeuAc), a sialic acid (SA), from CMP-SA to the non-reducing terminus of oligosaccharide chains of glycoconjugates. The addition of the sialic acid normally terminates oligosaccharide chain elongation except for polysialic chains found on neural cell adhesion molecule and gangliosides.
Known eukaryotic sialyltransferases involved in the synthesis of N- and O-glycan derivatives of the glycoprotien and glycolipid are summarized in Table 1, adapted from Palcic.sup.63. In the table, the R represents the remainder of the acceptor glycoprotein, glycolipid or oligosaccharide chain.
TABLE 1 EC sialyltransferase (SL) Number Linkage Synthesized Gal(2-6)-ST (ST6N) 2.4.99.1 NeuAc.alpha.2.fwdarw.6Gal.beta.1.fwdarw.4GlcNAc-R GalNAc.alpha.(2-6)-ST 2.4.99.4 NeuAC.alpha.2.fwdarw.6GalNAc.alpha.-R (ST6OI) Gal(2-3)-ST (ST30) 2.4.99.4 NeuAC.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4GalNAc.alpha.-R Gal(2-3)-ST (ST3N) 2.4.99.6 NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3/4GlcNAc-R GalNAc.alpha.(2-6)-ST 2.4.99.7 NeuAc.alpha.2.fwdarw.6 (ST6OII) .vertline. NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc-R N-Ac-neuramide .alpha.(2-8)- 2.4.99.8 NeuAc.alpha.2.fwdarw.8NeuAc.alpha.2.fwdarw.Gal.beta.-R sialyltransferase Gal.beta.1-3GlcNAc-ST NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GlcNAc-R
.alpha.2,3-sialyltransferases are useful eukaryotic enzymes for in vitro synthesis of N-linked and O-linked sialyl derivatives of glycoproteins, for determinations of acceptors, and other qualitative and quantitative research of glycoproteins. However, it was previously reported that 2,3-sialyltransferases would not synthesize N-linked and O-linked sialyl derivatives of glycoproteins or glycolipids where the acceptor glycoprotein or glycolipid possessed a fucosyl derivative in the penultimate position to the non-reducing sugar terminus of the oligosaccharide (U.S. Pat. No. 5,374,655.sup.67). This necessitated careful planning in the synthesis of certain fucosylated and sialylated oligosaccharides and in some cases required that certain steps be completed using chemical synthesis, rather than enzymatic synthesis.
In view of the above, it would be particularly advantageous to develop methods for the facile preparation of .alpha.-sialylated oligosaccharides from oligosaccharides having a fucosyl derivative in the penultimate position to the non-reducing sugar terminus of the oligosaccharide. The present invention accomplishes this by using an .alpha.2,3-sialyltransferase to effect efficient coupling of sialic acid activated as its CMP-nucleotide derivative (a donor saccharide) to a saccharide or an oligosaccharide having a fucosyl derivative in the penultimate position of the non-reducing end of the sugar moiety (acceptor oligosaccharide).